Journal of the American Association for Laboratory Animal Science Copyright 2014 by the American Association for Laboratory Animal Science
Vol 53, No 3 May 2014 Pages 267–272
Evaluation of the Use of Primate Undershirts as a Refinement Practice for Jacketed Rhesus Macaques (Macaca mulatta) Richard Kelly 3rd,1 Amy Carlson,1 Steven J Kern,2 Amy Field,1 Shannon Marko,1 Emily Bailey,1 Sarah Norris,2 Anna Honko,3 and Pedro Rico1 Jacketing of nonhuman primates (NHP) is a commonly used practice in the laboratory animal setting to support data collection with reduced direct human-to-animal interaction. NHP often wear jackets for several weeks, potentially leading to the formation of dermal lesions ranging from mild alopecia to severe full-thickness ulceration. We sought to evaluate the addition of a commercially available undershirt for primates as a possible refinement practice for our jacketed rhesus macaques. In this study, we compared the lesion count, location, and severity and differences in rectal body temperature between jacketed NHP with undershirts with those wearing the jackets alone. In both groups, most lesions (75%) were located at either the underarm or shoulder. The percentages of total lesions in the back and neck were lower in jacketed NHP that wore undershirts than in those that did not. In addition, the estimated odds of increased severity scores in jacketed NHP without undershirts was 1.80 times that for NHP that wore both jackets and undershirts. Both groups of NHP showed a significant decrease in dermal scores with time, indicating adaptation to the jackets with or without undershirts. However, there was no statistically significant decrease in lesion count, severity, or location in jacketed NHP that wore undershirts compared with those that did not. Abbreviations: CVC, central venous catheter; NHP, nonhuman primate; USAMRIID, United States Army Medical Research Institute for Infectious Diseases.
The principles of replacement, refinement, and reduction (also known as ‘the 3Rs’) were first described in 1959.3,12 These principles are now accepted and used widely throughout the animal research community.7 The most recent edition of the Guide for the Care and Use of Laboratory Animals defines the term ‘refinement’ as “modifications of husbandry or experimental procedures to enhance animal wellbeing and minimize or eliminate pain and distress.”8 Refinement encompasses a requirement to actively assess the adverse experimental effects of research and to implement control measures to reduce these effects.8,7,12 Two examples of refinement to obtain frequent phlebotomy samples or infusion of test samples in research animals without the use of anesthesia are the use of an indwelling central venous catheter (CVC) paired with a protective jacket and swiveltethering system and training nonhuman primates (NHP) to present extremities cageside for venipuncture.4,7,13 Physical contact is needed to acquire samples from trained NHP.4 At the United States Army Medical Research Institute for Infectious Diseases (USAMRIID), the majority of studies are conducted under ABSL3 or ABSL4 conditions, and physical contact with NHP is very limited due to the risk of occupational exposure. Therefore, USMARIID commonly uses CVC paired with jackets and a swivel-tethering system in research NHP. The CVC is surgically placed inside the NHP’s jugular vein or femoral vein and routed through subcutaneous tissue to exit the skin on the NHP’s back. The jacket is fitted on the NHP and serves as protective device that prohibits the NHP from removing the Received: 12 Sep 2013. Revision requested: 15 Oct 2013. Accepted: 28 Oct 2013. 1Veterinary Medicine, 2Statistics, and Virology, The United States Army Medical Research Institute for Infectious diseases, Fort Detrick Maryland. *Corresponding author. Email: [email protected]
indwelling catheter.2,3 When used alone, the jacket also can serve as a protective barrier for telemetry implants or to prevent self-injurious behavior.2 The swivel tether is a flexible stainless steel tube that connects the NHP’s jacket to the cage, thereby protecting the catheter as it exits the cage.3,13 The tether prevents specific postural changes and limits in-cage enhancements to those compatible with tethered animals.3,7,13 The combination of the 2 devices enables constant CVC access yet provides NHP with mobility (albeit limited) within the cage.3,13 As a result, this system eliminates risks due to physical contact or anesthetic restraint and improves personnel safety by decreasing the time that staff are in direct contact with NHP. Several companies make jackets for NHP, and available options for jacket use, design, and construction are extensive. For this study, we chose to use basic rhesus macaque jacket from Lomir Biomedical (Malone, NY). This jacket (Figure 1 upper left and right) has 2 layers of nylon mesh connected by a double-stitched strip of elastic material.9 It is constructed with heavy-duty zippers that can be secured with metal clips at the bottom of the jacket to deter manipulation by the NHP.9 The Lomir undershirt (Figure 1 lower left and right) is made from a spandex material that is flexible and form-fitting.9 Lomir markets the undershirt as a means for reducing rubbing and chafing when worn under the jacket.9 At USAMRIID, the jacket and swivel-tether system is often used in ABSL3 and ABSL4 infectious disease studies involving rhesus macaques (Macaca mulatta). Many containment studies at USAMRIID require jacketing of NHP for as long as 60 d, with an approximate timeline of 10 d for acclimation and surgery, 10 d for BSL4 acclimation, and 20 to 40 d for study duration. Although the use of the jacket and swivel-tether system decreases anesthetic variables and improves personnel safety, jacketed 267
jaalas13000131.indd 267
6/11/2014 9:35:02 AM
Vol 53, No 3 Journal of the American Association for Laboratory Animal Science May 2014
Figure 1. Rhesus macaque in a fitted jacket (upper left and right) and undershirt (lower left and right).
NHP sometimes develop dermal lesions of varying severity, due to excessive rubbing of the jacket against the animal’s skin.2 During prolonged studies, mild lesions (that is, those that show alopecia and some erythema and that are smaller than 1 cm in diameter)—if undiagnosed due to jacket coverage—can progress to become severe lesions (that is, with marked alopecia, erythema, and full-thickness ulceration and measuring more than 2 cm in diameter). In the research environment, it is not always appropriate to anesthetize animals for treatment because of physiologic effects and their relationship to research data.5,6 In some NHP infectious disease studies, increased core body temperature is the first indicator of disease.10 Researchers rely on published NHP data as a baseline to identify increases in an animal’s core body temperature.10 External factors that have the potential to increase core body temperature could confound research data. The Association of Primate Veterinarians has published the Guidelines for Jacket Use for Nonhuman Primates, which states “the jacket should not cause excessive rubbing, which may lead to skin lesions.”2 Various other publications also suggest the use of undershirts to reduce skin lesions.2,13 However, to date, no published studies have evaluated the common lesions associated with jackets in NHP, the benefits of using an undershirt to prevent these lesions, and the effects of jacket and undershirt placement on core body temperature. In the current study, we attempted to find a safe and practical solution to reduce or eliminate jacket lesions in NHP on long-term infectious disease studies. The objectives of our study were to evaluate the number, location, and severity of jacket lesions and to evaluate the use of
undershirts as a possible refinement practice for jacketed NHP. In addition, we wanted to determine whether using undershirts increased body temperature. We hypothesized that rhesus macaques in jackets and undershirts would have fewer, less severe lesions and a higher body temperature compared with those of rhesus macaques that wore jackets without undershirts.
Materials and Methods
Subjects. This study used 20 (10 female, 10 male) rhesus macaques (Macaca mulatta; age, 3.5 y to 7 y; weight, 4.8 to 7.8 kg). Each macaque was seronegative for Macacine herpesvirus 1, simian retrovirus type D, SIV, and simian T-lymphotropic leukemia virus. Macaques were tested twice annually for tuberculosis, and all remained negative. Research was conducted under an IACUC-approved protocol and in compliance with the Animal Welfare Act,1 PHS Policy,11 and other Federal statutes and regulations relating to animals and experiments involving animals. USAMRIID is accredited by AAALAC and adheres to principles stated in the Guide.8 For data application to ABSL3/4 environments, an exemption to full- or partial-contact housing of NHP was approved by the IACUC in light of the nature of diseases studied, the anticipated stress of frequent or permanent social separation from a cagemate, the current caging design used in containment, and for safety and sanitation concerns. Although NHP were not pair- or group-housed, they were housed in a room that allowed for visual, auditory, and olfactory contact of conspecifics. Enrichment was used to maximize other opportunities for NHP to partake in other species appropriate behaviors. The environment was
268
jaalas13000131.indd 268
6/11/2014 9:35:03 AM
Undershirts and jackets in rhesus macaques
maintained at 69 to 75 °F (22.6 to 23.9 °C), at a relative humidity of 30% to 70%, and on a 12:12-h light:dark cycle. The macaques were fed a commercial diet (2050 Teklad Global 20% Protein Primate Diet, Harlan Laboratories, Frederick, MD). Water was provided ad libitum. Various food and toy enrichments were used in the husbandry and care program. Health assessment. All NHP received baseline health assessments and behavioral assessments, which were conducted by a qualified veterinarian or veterinary technician 1 wk prior to study initiation. The baseline health assessment included a physical exam, CBC count, and serum chemistry. In addition, NHP were screened for any preexisting skin lesions and alopecia; no alopecia or skin lesions were noted prior to the start of study. Because this study was conducted in collaboration with a behavioral study, NHP behavior was monitored. Behavioral assessments included screening for preexisting abnormal behavior (that is, flipping, spinning, self-injurious behavior) and were performed prior to jacket placement, during the acclimation period, and daily throughout the course of the study. Any lesions resulting from sources other than jackets or undershirts were eliminated from data analysis. Grouping. On day 0, macaques underwent baseline evaluations as described previously. Immediately after the baseline evaluations, the macaques were matched by sex and weight (± 0.3 kg) for statistical purposes; one macaque from each statistical match was assigned randomly to wear a jacket only (that is, no undershirt; group A), whereas the other matched animal was fitted with both an undershirt and jacket (group B). Equipment and sizing. This study was conducted without the use of tether–swivel equipment. Equipment used in included primate jackets and undershirts (basic production models) manufactured by Lomir Biomedical (primate undershirts: size medium, catalog no. PJ 02CAB; size large, PJ 03CAB; and size extra-large, PJ 04CAB; primate jackets, size medium, catalog no. PJ 02; size large, PJ 03; and size extra-large, PJ 04). NHP were anesthetized with ketamine (3 to 10 mg/kg IM) and dexmedetomidine hydrochloride (0.02 to 0.05 mg/kg IM) and reversed with atipamezole hydrochloride (0.2 to 0.5 mg/kg IM). Hair was clipped from the waist to the neckline and from the arms to the point of the elbow, to replicate surgical preparation for CVC placement. The jacket chosen for each NHP was based on the manufacturer’s recommended weight ranges for rhesus macaques: NHP weighing 3 to 5 kg were fitted in medium jackets and undershirts; those weighing 5 to 7 kg received size large items; and macaques weighing 7 to 8 kg were fitted in extra-large jackets and undershirts. All macaques were measured to ensure that jackets and undershirts were fit according to manufacturer recommendations. According to the IACUC’s recommendation, NHP undershirts were placed inside out to prevent potential rubbing and chaffing from garment seams (Figure 1 lower left and right). Weekly physical exam and assessment. NHP were monitored once daily by veterinarians or qualified staff. During the 60-d study duration, macaques underwent physical assessment once each week. During each assessment, the macaques were anesthetized as previously described. Although NHP may not be routinely anesthetized during containments studies, the macaques on the current study were anesthetized weekly to increase data collection. Once the macaque was recumbent, a temperature was taken by using a rectal thermometer. The temperature was monitored to ensure that the addition of the jacket and undershirt did not adversely increase core body temperature, which might confound infectious disease data. A 4-mL blood sample was obtained from a peripheral vessel for
CBC counts and serum chemistry evaluation. Garments then were removed and examined for cleanliness and damage. If garments were soiled or had excessive damage, they were replaced. A complete physical exam was performed. According to physical exam findings, each macaque received a dermal score that was determined by multiplying the lesion severity by the lesion diameter. Each week, all lesions present received a dermal score. All scoring was performed by trained veterinarians. Each score was reviewed and discussed among the trained observers, to decrease variability. The lesion severity was scored as 0 for no lesions observed, 1 for erythema without ulceration, 2 for erythema with mild to moderate ulceration, and 3 for alopecia and erythema with full-thickness ulceration. The lesion size was scored as 1 for a diameter of less than 1 cm, 2 for a diameter between 1 and 2 cm, and 3 for a diameter greater than 2 cm. Any lesions with a severity of 3 were cleaned and medicated with triple-antibiotic ointment and monitored for signs of infection. Lesion location was assessed according to 7 main areas: arms, back, chest, neck, shoulders, underarms, and waist. Statistical analysis. The objective of the statistical analysis was to compare mean lesion location, lesion count, dermal score, and rectal temperature between rhesus macaques wearing jackets only and those wearing jackets and undershirts over 60 d of observation. Outcome variables were analyzed with linear and generalized linear models using the same format of fixed group and time, group×time interaction effects, and a random animal effect to account for repeated measures. Lesion count data were analyzed by using zero-inflated Poisson regressions. Zero-inflated Poisson regressions were used instead of traditional Poisson regressions or negative binomial regressions because of the high propensity of observations with skin lesion scores of 0. Dermal scores were analyzed by using nonparametric repeated-measures ANOVA and replacing raw dermal scores with ranks. Body temperature was analyzed by using repeated-measures ANOVA. Lesion severity scores were analyzed by using an ordinal multinomial logistic regression. All statistical analysis was performed by using SAS version 9.3 (SAS Institute, Cary, NC). All hypothesis tests were performed with 2-sided alternatives and were considered significant at α = 0.05.
Results
All macaques successfully acclimated to jackets after a behavioral screening period of 1 wk, consistent with published criteria.2 Of the 20 NHP jacketed, 19 remained on study to the conclusion of data collection; the remaining NHP was removed from the study at week 6 due to the macaque’s persistent removal of the jacket. Lesion counts in each body location were analyzed by using a zero-inflated Poisson regression with repeated measures (Table 1). Lesion counts were obtained during the weekly dermal lesion assessments (Figure 2). Statistical analysis revealed no significant difference in mean lesion count between groups (–0.53 lesions; 95% CI, −1.85 to 0.79; P = 0.4101). There was no statistically significant interaction between group and time (0.03 lesions per week; 95% CI, −0.11 to 0.17; P = 0.6287). On average, there was a small but significant negative effect of time over the course of the experiment (−0.15 lesions per week; 95% CI, −0.25 to −0.050; P = 0.0074), suggesting that macaques in both groups gradually had fewer lesions over time. The distribution of lesion location between groups was similar. More than 50% of lesions were located at underarms (Figure 3), and more than 75% of all lesions were at the underarm or shoulder. However, macaques that wore undershirts beneath jackets (group B) had a smaller percentage of lesions on the back 269
jaalas13000131.indd 269
6/11/2014 9:35:04 AM
Vol 53, No 3 Journal of the American Association for Laboratory Animal Science May 2014
Table 1. Summary of analysis of lesion data from macaques wearing jackets without (group A) or with (group B) undershirts No. of lesions Odds ratio estimate ± SE
Group A (n = 111)
P
Group B (n = 82)
Arm
0.02 ± 0.02
Back
0.26 ± 0.09
0.3229
1
0
0.0062
13
1
Chest
–0.06 ± 0.04
Neck
0.15 ± 0.09
0.0905
0
3
0.0779
11
Shoulder
4
0.17 ± 0.18
0.3405
29
20
Underarm
0.06 ± 0.21
0.7866
55
54
Waist
0.04 ± 0.03
0.1634
2
0
Location
Figure 2. Lesion counts over 60 d for macaques wearing jackets without (group A) or with (group B) undershirts.
(1.2%) and neck (4.9%) than did those that wore jackets (group A; 11.7% and 9.9%, respectively; Figure 4). Lesion severity was recorded as an ordinal variable with a value of 1, 2, or 3, indicating increasing severity. The severity score outcome was modeled as an ordinal multinomial logistic regression against the treatment group variable. The estimated odds of higher severity scores in group A (macaques with jackets only) than in group B (macaques with undershirts and jackets) was 1.80 times the odds of higher severity scores in group B than in group A (95% CI, 0.71 to 4.53; P = 0.2150). In addition, 18% of lesions in group A and 11% of lesions in group B had a severity score of 2 or greater. One lesion in group A achieved a severity score of 3 (Figure 3 B) and received medical treatment. Group had no significant effect on the odds of having a lower dermal score (odds ratio = 1.27; 95% CI, 0.11 to 15.20; P = 0.8485). However time did significantly affect the odds of having a lower dermal score (odds ratio, 1.34; 95% CI, 1.15 to 1.55; P = 0.0002); that is, the odds of having a lower dermal score during the current week was 1.34 times that of having a lower dermal score during the previous week. No significant interaction was found between group and week (odds ratio, 1.25; 95% CI, 0.93 to 1.68; P = 0.1401). Average rectal temperature did not differ significantly between treatment groups (mean difference, 0.001 °F; 95% CI, −0.66 to 0.66; P = 0.9969), nor was any interaction between group
and week observed (mean difference, 0.05 °F per week; 95% CI, −0.05 to 0.14; P = 0.3148). A small but significant negative effect of time was present (mean difference, −0.07 °F per week; 95% CI, −0.05 to 0.14; P = 0.0054), suggesting a slight decrease in body temperature over the course of the experiment (Figure 5).
Discussion
Jackets for NHP are commonly used in laboratory research. The NHP jacket is a garment that protects telemetry devices and CVC and provides barrier protection for medical treatments.2,3,9,13 When paired with tether equipment, jacketing is a refinement practice that affords NHP a limited range of movement as it gives scientists access to CVC from outside the cage and without the need for anesthesia.3,13 Over time, jackets can cause dermal lesions.2,13 In the current study, NHP undershirts were evaluated as possible solutions to reduce or eliminate dermal lesions due to jackets. Lesions in NHP wearing jackets and undershirts were compared with those in NHP wearing jackets only throughout a 60-d study duration. The results of the current study demonstrated that the mean lesion count in macaques that wore both jackets and undershirts (group B) was not significantly different from that of macaques that wore jackets only (group A). In both groups, time was significantly associated with a moderate reduction in mean lesion count, fewer lesions, and lower dermal score. The results suggest that lesions did not reoccur after the primary lesions healed. The undershirt did not significantly affect the development of lesions or the number of lesions. This outcome can be explained by the fact that the jackets and undershirts caused primary lesions early in the study; these lesions healed by second intention over time, with the formation of calluses. We did not perform a histologic skin evaluation for calluses, which could be beneficial in a future study. In addition, the upper body of all macaques was shaved prior to jacket placement to simulate studies involving CVC placement. Over time, the hair grew back and may have provided protection from the jacket or undershirt. The shaving may have had a negative effect on the measured parameters, and a more localized shaving technique may provide better dermal protection. The majority of lesions in both groups occurred in the underarm area, with the shoulder as the second most-common area. Jackets caused fewer lesions in the neck area and on the back. Overall, the undershirt did not provide a significant protection. The majority of lesions were characterized by mild erythema (Figure 3 A). Jackets rarely caused lesions that required treatment. The jackets used in this experiment were the most basic version offered by the manufacturer. Other configurations with more padding and specific customization according to customer need and individual NHP measurements are available commercially. Jacket systems that offer more padding and elasticity under arms and in the shoulder area may be beneficial. For sizing and study-specific garments, custom measurements and configurations can be submitted to the manufacturer. During infectious disease studies, body temperature is often used as an indicator of disease. We therefore measured the rectal temperature of our macaques to determine whether the undershirt significantly increased core body temperature. No significant difference was observed between treatment groups. However, a small but significant negative effect of time was observed, suggesting that fitting NHP with jackets with or without undershirts would not confound the temperature data often used to monitor NHP disease progression. Collectively, our current study evaluated NHP undershirts— placed inside out—as a possible refinement practice for jacketed
270
jaalas13000131.indd 270
6/11/2014 9:35:04 AM
Undershirts and jackets in rhesus macaques
Figure 3. Garment-induced dermal lesions in the underarm area of a macaque.
Figure 4. Percentage of lesions in each body location (AR, arms; BK, back; CH, chest; NK, neck; SH, shoulders; UA, underarm; W, waist) for macaques wearing jackets without (group A) or with (group B) undershirts.
rhesus macaques. Group differences in lesion count, severity, temperature, and location were not significant. We did not find any disadvantages associated with using undershirts under jackets. When NHP jackets and undershirts are used, research staff must be trained appropriately in fitting NHP garments, and sizing choices should be confirmed with the manufacturer. In addition, NHP should be monitored routinely for adverse clinical signs. This study was conducted under ABSL2 conditions without the use of swivel–tether equipment. A similar study using such equipment may yield different data concerning lesion location, count, and severity. In addition, a study conducted in conjunction with an ABSL4 study may more closely replicate conditions and limitations of animals on infectious disease studies. In addition, a study comparing different study durations might provide valuable data.
Acknowledgments
Figure 5. Rectal body temperatures in macaques wearing jackets without (group A) or with (group B) undershirts over the 60-d observational period.
We thank Dr Krystal Bean, Dr Joanna Fishback, Dr Nancy Keller, Dr Alec Hail, Dr Susan Goodwin, SGT Michael Lavorgna, SGT Stephen Pounders, SPC Michele Johnson, Charity Stagg, and the support staff of the Veterinary Medicine Division at USAMRIID. The opinions, interpretations, conclusions, and recommendations are those of the author and are not necessarily endorsed by the US Army. 271
jaalas13000131.indd 271
6/11/2014 9:35:07 AM
Vol 53, No 3 Journal of the American Association for Laboratory Animal Science May 2014
References 1. Animal Welfare Act as Amended. 2008. 7 USC §2131–2159. 2. Association of Primate Veterinarians. [Internet]. 2013. Guidelines for jacket use for nonhuman primates. [Cited 17 October 2013]. Available at: http://www.primatevets.org/Content/files/Public/ education/NHP_Jacket_Use_Guidelines.pdf 3. Chatham AK. 1985. Jacket-and-swivel tethering systems. Lab Animal 14:29–33. 4. Coleman K, Pranger L, Maier A. 2008. Training rhesus macaques for venipuncture using a positive-reinforcement technique: a comparison with chimpanzees. J Am Assoc Lab Anim Sci 47:37–41. 5. Foster CD, Hunter TC, Gibbs PH, Leffel EK. 2008. Whole-body plethysmography in African green monkeys (Chlorocebus aethiops) with and without jackets. J Am Assoc Lab Anim Sci 47:52–55. 6. Gamble CS, Jacobsen KO, Leffel EK. 2007. Use of a low-concentration heparin solution to extend the life of central venous catheters in African green monkeys (Cholorocebus aethiops). J Am Assoc Lab Anim Sci 46:58–60. 7. Guhad F. 2005. Introduction to the 3Rs (refinement, reduction, and replacement). Contemp Top Lab Anim Sci 44:58–59.
8. Institute for Laboratory Animal Research. 2011. Guide for the care and use of laboratory animals, 8th ed. Washington (DC): National Academies Press. 9. Lomir Biomedical. [Internet] 2013. Jackets and undershirts. [Cited 17 October 2013]. Available at: http://lomir.com/productsservices/jackets/jackets-undershirts/ 10. Nelson M, Lever MS, Savage VL. 2009. Establishment of lethal inhalational infection with Francisella tularensis (tularaemia) in the common marmoset (Callithrix jacchus). Int J Exp Pathol 90:109–118. 11. Office of Laboratory Animal Welfare. [Internet]. 2002. Public health service policy on humane care and use of laboratory animals. [Cited 17 October 2013]. Available at: http://grants.nih.gov/ grants/olaw/references/phspol.htm 12. Russell WMS, Burch RL. 1959. The principles of humane experimental technique. London (UK): Methuen. 13. Turner PV, Pekow C, Vasbinder MA. 2011. Administration of substances to laboratory animals; equipment considerations, vehicle selection, and solute preparation. J Am Assoc Lab Anim Sci 50:614–627.
272
jaalas13000131.indd 272
6/11/2014 9:35:07 AM
Vol 53, No 3 May 2014 Pages 267–272
Evaluation of the Use of Primate Undershirts as a Refinement Practice for Jacketed Rhesus Macaques (Macaca mulatta) Richard Kelly 3rd,1 Amy Carlson,1 Steven J Kern,2 Amy Field,1 Shannon Marko,1 Emily Bailey,1 Sarah Norris,2 Anna Honko,3 and Pedro Rico1 Jacketing of nonhuman primates (NHP) is a commonly used practice in the laboratory animal setting to support data collection with reduced direct human-to-animal interaction. NHP often wear jackets for several weeks, potentially leading to the formation of dermal lesions ranging from mild alopecia to severe full-thickness ulceration. We sought to evaluate the addition of a commercially available undershirt for primates as a possible refinement practice for our jacketed rhesus macaques. In this study, we compared the lesion count, location, and severity and differences in rectal body temperature between jacketed NHP with undershirts with those wearing the jackets alone. In both groups, most lesions (75%) were located at either the underarm or shoulder. The percentages of total lesions in the back and neck were lower in jacketed NHP that wore undershirts than in those that did not. In addition, the estimated odds of increased severity scores in jacketed NHP without undershirts was 1.80 times that for NHP that wore both jackets and undershirts. Both groups of NHP showed a significant decrease in dermal scores with time, indicating adaptation to the jackets with or without undershirts. However, there was no statistically significant decrease in lesion count, severity, or location in jacketed NHP that wore undershirts compared with those that did not. Abbreviations: CVC, central venous catheter; NHP, nonhuman primate; USAMRIID, United States Army Medical Research Institute for Infectious Diseases.
The principles of replacement, refinement, and reduction (also known as ‘the 3Rs’) were first described in 1959.3,12 These principles are now accepted and used widely throughout the animal research community.7 The most recent edition of the Guide for the Care and Use of Laboratory Animals defines the term ‘refinement’ as “modifications of husbandry or experimental procedures to enhance animal wellbeing and minimize or eliminate pain and distress.”8 Refinement encompasses a requirement to actively assess the adverse experimental effects of research and to implement control measures to reduce these effects.8,7,12 Two examples of refinement to obtain frequent phlebotomy samples or infusion of test samples in research animals without the use of anesthesia are the use of an indwelling central venous catheter (CVC) paired with a protective jacket and swiveltethering system and training nonhuman primates (NHP) to present extremities cageside for venipuncture.4,7,13 Physical contact is needed to acquire samples from trained NHP.4 At the United States Army Medical Research Institute for Infectious Diseases (USAMRIID), the majority of studies are conducted under ABSL3 or ABSL4 conditions, and physical contact with NHP is very limited due to the risk of occupational exposure. Therefore, USMARIID commonly uses CVC paired with jackets and a swivel-tethering system in research NHP. The CVC is surgically placed inside the NHP’s jugular vein or femoral vein and routed through subcutaneous tissue to exit the skin on the NHP’s back. The jacket is fitted on the NHP and serves as protective device that prohibits the NHP from removing the Received: 12 Sep 2013. Revision requested: 15 Oct 2013. Accepted: 28 Oct 2013. 1Veterinary Medicine, 2Statistics, and Virology, The United States Army Medical Research Institute for Infectious diseases, Fort Detrick Maryland. *Corresponding author. Email: [email protected]
indwelling catheter.2,3 When used alone, the jacket also can serve as a protective barrier for telemetry implants or to prevent self-injurious behavior.2 The swivel tether is a flexible stainless steel tube that connects the NHP’s jacket to the cage, thereby protecting the catheter as it exits the cage.3,13 The tether prevents specific postural changes and limits in-cage enhancements to those compatible with tethered animals.3,7,13 The combination of the 2 devices enables constant CVC access yet provides NHP with mobility (albeit limited) within the cage.3,13 As a result, this system eliminates risks due to physical contact or anesthetic restraint and improves personnel safety by decreasing the time that staff are in direct contact with NHP. Several companies make jackets for NHP, and available options for jacket use, design, and construction are extensive. For this study, we chose to use basic rhesus macaque jacket from Lomir Biomedical (Malone, NY). This jacket (Figure 1 upper left and right) has 2 layers of nylon mesh connected by a double-stitched strip of elastic material.9 It is constructed with heavy-duty zippers that can be secured with metal clips at the bottom of the jacket to deter manipulation by the NHP.9 The Lomir undershirt (Figure 1 lower left and right) is made from a spandex material that is flexible and form-fitting.9 Lomir markets the undershirt as a means for reducing rubbing and chafing when worn under the jacket.9 At USAMRIID, the jacket and swivel-tether system is often used in ABSL3 and ABSL4 infectious disease studies involving rhesus macaques (Macaca mulatta). Many containment studies at USAMRIID require jacketing of NHP for as long as 60 d, with an approximate timeline of 10 d for acclimation and surgery, 10 d for BSL4 acclimation, and 20 to 40 d for study duration. Although the use of the jacket and swivel-tether system decreases anesthetic variables and improves personnel safety, jacketed 267
jaalas13000131.indd 267
6/11/2014 9:35:02 AM
Vol 53, No 3 Journal of the American Association for Laboratory Animal Science May 2014
Figure 1. Rhesus macaque in a fitted jacket (upper left and right) and undershirt (lower left and right).
NHP sometimes develop dermal lesions of varying severity, due to excessive rubbing of the jacket against the animal’s skin.2 During prolonged studies, mild lesions (that is, those that show alopecia and some erythema and that are smaller than 1 cm in diameter)—if undiagnosed due to jacket coverage—can progress to become severe lesions (that is, with marked alopecia, erythema, and full-thickness ulceration and measuring more than 2 cm in diameter). In the research environment, it is not always appropriate to anesthetize animals for treatment because of physiologic effects and their relationship to research data.5,6 In some NHP infectious disease studies, increased core body temperature is the first indicator of disease.10 Researchers rely on published NHP data as a baseline to identify increases in an animal’s core body temperature.10 External factors that have the potential to increase core body temperature could confound research data. The Association of Primate Veterinarians has published the Guidelines for Jacket Use for Nonhuman Primates, which states “the jacket should not cause excessive rubbing, which may lead to skin lesions.”2 Various other publications also suggest the use of undershirts to reduce skin lesions.2,13 However, to date, no published studies have evaluated the common lesions associated with jackets in NHP, the benefits of using an undershirt to prevent these lesions, and the effects of jacket and undershirt placement on core body temperature. In the current study, we attempted to find a safe and practical solution to reduce or eliminate jacket lesions in NHP on long-term infectious disease studies. The objectives of our study were to evaluate the number, location, and severity of jacket lesions and to evaluate the use of
undershirts as a possible refinement practice for jacketed NHP. In addition, we wanted to determine whether using undershirts increased body temperature. We hypothesized that rhesus macaques in jackets and undershirts would have fewer, less severe lesions and a higher body temperature compared with those of rhesus macaques that wore jackets without undershirts.
Materials and Methods
Subjects. This study used 20 (10 female, 10 male) rhesus macaques (Macaca mulatta; age, 3.5 y to 7 y; weight, 4.8 to 7.8 kg). Each macaque was seronegative for Macacine herpesvirus 1, simian retrovirus type D, SIV, and simian T-lymphotropic leukemia virus. Macaques were tested twice annually for tuberculosis, and all remained negative. Research was conducted under an IACUC-approved protocol and in compliance with the Animal Welfare Act,1 PHS Policy,11 and other Federal statutes and regulations relating to animals and experiments involving animals. USAMRIID is accredited by AAALAC and adheres to principles stated in the Guide.8 For data application to ABSL3/4 environments, an exemption to full- or partial-contact housing of NHP was approved by the IACUC in light of the nature of diseases studied, the anticipated stress of frequent or permanent social separation from a cagemate, the current caging design used in containment, and for safety and sanitation concerns. Although NHP were not pair- or group-housed, they were housed in a room that allowed for visual, auditory, and olfactory contact of conspecifics. Enrichment was used to maximize other opportunities for NHP to partake in other species appropriate behaviors. The environment was
268
jaalas13000131.indd 268
6/11/2014 9:35:03 AM
Undershirts and jackets in rhesus macaques
maintained at 69 to 75 °F (22.6 to 23.9 °C), at a relative humidity of 30% to 70%, and on a 12:12-h light:dark cycle. The macaques were fed a commercial diet (2050 Teklad Global 20% Protein Primate Diet, Harlan Laboratories, Frederick, MD). Water was provided ad libitum. Various food and toy enrichments were used in the husbandry and care program. Health assessment. All NHP received baseline health assessments and behavioral assessments, which were conducted by a qualified veterinarian or veterinary technician 1 wk prior to study initiation. The baseline health assessment included a physical exam, CBC count, and serum chemistry. In addition, NHP were screened for any preexisting skin lesions and alopecia; no alopecia or skin lesions were noted prior to the start of study. Because this study was conducted in collaboration with a behavioral study, NHP behavior was monitored. Behavioral assessments included screening for preexisting abnormal behavior (that is, flipping, spinning, self-injurious behavior) and were performed prior to jacket placement, during the acclimation period, and daily throughout the course of the study. Any lesions resulting from sources other than jackets or undershirts were eliminated from data analysis. Grouping. On day 0, macaques underwent baseline evaluations as described previously. Immediately after the baseline evaluations, the macaques were matched by sex and weight (± 0.3 kg) for statistical purposes; one macaque from each statistical match was assigned randomly to wear a jacket only (that is, no undershirt; group A), whereas the other matched animal was fitted with both an undershirt and jacket (group B). Equipment and sizing. This study was conducted without the use of tether–swivel equipment. Equipment used in included primate jackets and undershirts (basic production models) manufactured by Lomir Biomedical (primate undershirts: size medium, catalog no. PJ 02CAB; size large, PJ 03CAB; and size extra-large, PJ 04CAB; primate jackets, size medium, catalog no. PJ 02; size large, PJ 03; and size extra-large, PJ 04). NHP were anesthetized with ketamine (3 to 10 mg/kg IM) and dexmedetomidine hydrochloride (0.02 to 0.05 mg/kg IM) and reversed with atipamezole hydrochloride (0.2 to 0.5 mg/kg IM). Hair was clipped from the waist to the neckline and from the arms to the point of the elbow, to replicate surgical preparation for CVC placement. The jacket chosen for each NHP was based on the manufacturer’s recommended weight ranges for rhesus macaques: NHP weighing 3 to 5 kg were fitted in medium jackets and undershirts; those weighing 5 to 7 kg received size large items; and macaques weighing 7 to 8 kg were fitted in extra-large jackets and undershirts. All macaques were measured to ensure that jackets and undershirts were fit according to manufacturer recommendations. According to the IACUC’s recommendation, NHP undershirts were placed inside out to prevent potential rubbing and chaffing from garment seams (Figure 1 lower left and right). Weekly physical exam and assessment. NHP were monitored once daily by veterinarians or qualified staff. During the 60-d study duration, macaques underwent physical assessment once each week. During each assessment, the macaques were anesthetized as previously described. Although NHP may not be routinely anesthetized during containments studies, the macaques on the current study were anesthetized weekly to increase data collection. Once the macaque was recumbent, a temperature was taken by using a rectal thermometer. The temperature was monitored to ensure that the addition of the jacket and undershirt did not adversely increase core body temperature, which might confound infectious disease data. A 4-mL blood sample was obtained from a peripheral vessel for
CBC counts and serum chemistry evaluation. Garments then were removed and examined for cleanliness and damage. If garments were soiled or had excessive damage, they were replaced. A complete physical exam was performed. According to physical exam findings, each macaque received a dermal score that was determined by multiplying the lesion severity by the lesion diameter. Each week, all lesions present received a dermal score. All scoring was performed by trained veterinarians. Each score was reviewed and discussed among the trained observers, to decrease variability. The lesion severity was scored as 0 for no lesions observed, 1 for erythema without ulceration, 2 for erythema with mild to moderate ulceration, and 3 for alopecia and erythema with full-thickness ulceration. The lesion size was scored as 1 for a diameter of less than 1 cm, 2 for a diameter between 1 and 2 cm, and 3 for a diameter greater than 2 cm. Any lesions with a severity of 3 were cleaned and medicated with triple-antibiotic ointment and monitored for signs of infection. Lesion location was assessed according to 7 main areas: arms, back, chest, neck, shoulders, underarms, and waist. Statistical analysis. The objective of the statistical analysis was to compare mean lesion location, lesion count, dermal score, and rectal temperature between rhesus macaques wearing jackets only and those wearing jackets and undershirts over 60 d of observation. Outcome variables were analyzed with linear and generalized linear models using the same format of fixed group and time, group×time interaction effects, and a random animal effect to account for repeated measures. Lesion count data were analyzed by using zero-inflated Poisson regressions. Zero-inflated Poisson regressions were used instead of traditional Poisson regressions or negative binomial regressions because of the high propensity of observations with skin lesion scores of 0. Dermal scores were analyzed by using nonparametric repeated-measures ANOVA and replacing raw dermal scores with ranks. Body temperature was analyzed by using repeated-measures ANOVA. Lesion severity scores were analyzed by using an ordinal multinomial logistic regression. All statistical analysis was performed by using SAS version 9.3 (SAS Institute, Cary, NC). All hypothesis tests were performed with 2-sided alternatives and were considered significant at α = 0.05.
Results
All macaques successfully acclimated to jackets after a behavioral screening period of 1 wk, consistent with published criteria.2 Of the 20 NHP jacketed, 19 remained on study to the conclusion of data collection; the remaining NHP was removed from the study at week 6 due to the macaque’s persistent removal of the jacket. Lesion counts in each body location were analyzed by using a zero-inflated Poisson regression with repeated measures (Table 1). Lesion counts were obtained during the weekly dermal lesion assessments (Figure 2). Statistical analysis revealed no significant difference in mean lesion count between groups (–0.53 lesions; 95% CI, −1.85 to 0.79; P = 0.4101). There was no statistically significant interaction between group and time (0.03 lesions per week; 95% CI, −0.11 to 0.17; P = 0.6287). On average, there was a small but significant negative effect of time over the course of the experiment (−0.15 lesions per week; 95% CI, −0.25 to −0.050; P = 0.0074), suggesting that macaques in both groups gradually had fewer lesions over time. The distribution of lesion location between groups was similar. More than 50% of lesions were located at underarms (Figure 3), and more than 75% of all lesions were at the underarm or shoulder. However, macaques that wore undershirts beneath jackets (group B) had a smaller percentage of lesions on the back 269
jaalas13000131.indd 269
6/11/2014 9:35:04 AM
Vol 53, No 3 Journal of the American Association for Laboratory Animal Science May 2014
Table 1. Summary of analysis of lesion data from macaques wearing jackets without (group A) or with (group B) undershirts No. of lesions Odds ratio estimate ± SE
Group A (n = 111)
P
Group B (n = 82)
Arm
0.02 ± 0.02
Back
0.26 ± 0.09
0.3229
1
0
0.0062
13
1
Chest
–0.06 ± 0.04
Neck
0.15 ± 0.09
0.0905
0
3
0.0779
11
Shoulder
4
0.17 ± 0.18
0.3405
29
20
Underarm
0.06 ± 0.21
0.7866
55
54
Waist
0.04 ± 0.03
0.1634
2
0
Location
Figure 2. Lesion counts over 60 d for macaques wearing jackets without (group A) or with (group B) undershirts.
(1.2%) and neck (4.9%) than did those that wore jackets (group A; 11.7% and 9.9%, respectively; Figure 4). Lesion severity was recorded as an ordinal variable with a value of 1, 2, or 3, indicating increasing severity. The severity score outcome was modeled as an ordinal multinomial logistic regression against the treatment group variable. The estimated odds of higher severity scores in group A (macaques with jackets only) than in group B (macaques with undershirts and jackets) was 1.80 times the odds of higher severity scores in group B than in group A (95% CI, 0.71 to 4.53; P = 0.2150). In addition, 18% of lesions in group A and 11% of lesions in group B had a severity score of 2 or greater. One lesion in group A achieved a severity score of 3 (Figure 3 B) and received medical treatment. Group had no significant effect on the odds of having a lower dermal score (odds ratio = 1.27; 95% CI, 0.11 to 15.20; P = 0.8485). However time did significantly affect the odds of having a lower dermal score (odds ratio, 1.34; 95% CI, 1.15 to 1.55; P = 0.0002); that is, the odds of having a lower dermal score during the current week was 1.34 times that of having a lower dermal score during the previous week. No significant interaction was found between group and week (odds ratio, 1.25; 95% CI, 0.93 to 1.68; P = 0.1401). Average rectal temperature did not differ significantly between treatment groups (mean difference, 0.001 °F; 95% CI, −0.66 to 0.66; P = 0.9969), nor was any interaction between group
and week observed (mean difference, 0.05 °F per week; 95% CI, −0.05 to 0.14; P = 0.3148). A small but significant negative effect of time was present (mean difference, −0.07 °F per week; 95% CI, −0.05 to 0.14; P = 0.0054), suggesting a slight decrease in body temperature over the course of the experiment (Figure 5).
Discussion
Jackets for NHP are commonly used in laboratory research. The NHP jacket is a garment that protects telemetry devices and CVC and provides barrier protection for medical treatments.2,3,9,13 When paired with tether equipment, jacketing is a refinement practice that affords NHP a limited range of movement as it gives scientists access to CVC from outside the cage and without the need for anesthesia.3,13 Over time, jackets can cause dermal lesions.2,13 In the current study, NHP undershirts were evaluated as possible solutions to reduce or eliminate dermal lesions due to jackets. Lesions in NHP wearing jackets and undershirts were compared with those in NHP wearing jackets only throughout a 60-d study duration. The results of the current study demonstrated that the mean lesion count in macaques that wore both jackets and undershirts (group B) was not significantly different from that of macaques that wore jackets only (group A). In both groups, time was significantly associated with a moderate reduction in mean lesion count, fewer lesions, and lower dermal score. The results suggest that lesions did not reoccur after the primary lesions healed. The undershirt did not significantly affect the development of lesions or the number of lesions. This outcome can be explained by the fact that the jackets and undershirts caused primary lesions early in the study; these lesions healed by second intention over time, with the formation of calluses. We did not perform a histologic skin evaluation for calluses, which could be beneficial in a future study. In addition, the upper body of all macaques was shaved prior to jacket placement to simulate studies involving CVC placement. Over time, the hair grew back and may have provided protection from the jacket or undershirt. The shaving may have had a negative effect on the measured parameters, and a more localized shaving technique may provide better dermal protection. The majority of lesions in both groups occurred in the underarm area, with the shoulder as the second most-common area. Jackets caused fewer lesions in the neck area and on the back. Overall, the undershirt did not provide a significant protection. The majority of lesions were characterized by mild erythema (Figure 3 A). Jackets rarely caused lesions that required treatment. The jackets used in this experiment were the most basic version offered by the manufacturer. Other configurations with more padding and specific customization according to customer need and individual NHP measurements are available commercially. Jacket systems that offer more padding and elasticity under arms and in the shoulder area may be beneficial. For sizing and study-specific garments, custom measurements and configurations can be submitted to the manufacturer. During infectious disease studies, body temperature is often used as an indicator of disease. We therefore measured the rectal temperature of our macaques to determine whether the undershirt significantly increased core body temperature. No significant difference was observed between treatment groups. However, a small but significant negative effect of time was observed, suggesting that fitting NHP with jackets with or without undershirts would not confound the temperature data often used to monitor NHP disease progression. Collectively, our current study evaluated NHP undershirts— placed inside out—as a possible refinement practice for jacketed
270
jaalas13000131.indd 270
6/11/2014 9:35:04 AM
Undershirts and jackets in rhesus macaques
Figure 3. Garment-induced dermal lesions in the underarm area of a macaque.
Figure 4. Percentage of lesions in each body location (AR, arms; BK, back; CH, chest; NK, neck; SH, shoulders; UA, underarm; W, waist) for macaques wearing jackets without (group A) or with (group B) undershirts.
rhesus macaques. Group differences in lesion count, severity, temperature, and location were not significant. We did not find any disadvantages associated with using undershirts under jackets. When NHP jackets and undershirts are used, research staff must be trained appropriately in fitting NHP garments, and sizing choices should be confirmed with the manufacturer. In addition, NHP should be monitored routinely for adverse clinical signs. This study was conducted under ABSL2 conditions without the use of swivel–tether equipment. A similar study using such equipment may yield different data concerning lesion location, count, and severity. In addition, a study conducted in conjunction with an ABSL4 study may more closely replicate conditions and limitations of animals on infectious disease studies. In addition, a study comparing different study durations might provide valuable data.
Acknowledgments
Figure 5. Rectal body temperatures in macaques wearing jackets without (group A) or with (group B) undershirts over the 60-d observational period.
We thank Dr Krystal Bean, Dr Joanna Fishback, Dr Nancy Keller, Dr Alec Hail, Dr Susan Goodwin, SGT Michael Lavorgna, SGT Stephen Pounders, SPC Michele Johnson, Charity Stagg, and the support staff of the Veterinary Medicine Division at USAMRIID. The opinions, interpretations, conclusions, and recommendations are those of the author and are not necessarily endorsed by the US Army. 271
jaalas13000131.indd 271
6/11/2014 9:35:07 AM
Vol 53, No 3 Journal of the American Association for Laboratory Animal Science May 2014
References 1. Animal Welfare Act as Amended. 2008. 7 USC §2131–2159. 2. Association of Primate Veterinarians. [Internet]. 2013. Guidelines for jacket use for nonhuman primates. [Cited 17 October 2013]. Available at: http://www.primatevets.org/Content/files/Public/ education/NHP_Jacket_Use_Guidelines.pdf 3. Chatham AK. 1985. Jacket-and-swivel tethering systems. Lab Animal 14:29–33. 4. Coleman K, Pranger L, Maier A. 2008. Training rhesus macaques for venipuncture using a positive-reinforcement technique: a comparison with chimpanzees. J Am Assoc Lab Anim Sci 47:37–41. 5. Foster CD, Hunter TC, Gibbs PH, Leffel EK. 2008. Whole-body plethysmography in African green monkeys (Chlorocebus aethiops) with and without jackets. J Am Assoc Lab Anim Sci 47:52–55. 6. Gamble CS, Jacobsen KO, Leffel EK. 2007. Use of a low-concentration heparin solution to extend the life of central venous catheters in African green monkeys (Cholorocebus aethiops). J Am Assoc Lab Anim Sci 46:58–60. 7. Guhad F. 2005. Introduction to the 3Rs (refinement, reduction, and replacement). Contemp Top Lab Anim Sci 44:58–59.
8. Institute for Laboratory Animal Research. 2011. Guide for the care and use of laboratory animals, 8th ed. Washington (DC): National Academies Press. 9. Lomir Biomedical. [Internet] 2013. Jackets and undershirts. [Cited 17 October 2013]. Available at: http://lomir.com/productsservices/jackets/jackets-undershirts/ 10. Nelson M, Lever MS, Savage VL. 2009. Establishment of lethal inhalational infection with Francisella tularensis (tularaemia) in the common marmoset (Callithrix jacchus). Int J Exp Pathol 90:109–118. 11. Office of Laboratory Animal Welfare. [Internet]. 2002. Public health service policy on humane care and use of laboratory animals. [Cited 17 October 2013]. Available at: http://grants.nih.gov/ grants/olaw/references/phspol.htm 12. Russell WMS, Burch RL. 1959. The principles of humane experimental technique. London (UK): Methuen. 13. Turner PV, Pekow C, Vasbinder MA. 2011. Administration of substances to laboratory animals; equipment considerations, vehicle selection, and solute preparation. J Am Assoc Lab Anim Sci 50:614–627.
272
jaalas13000131.indd 272
6/11/2014 9:35:07 AM
